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Bc34lec3 Nucleic Acids

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    Inhibitors of the Genetic

    Mechanisms

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    Macrolides

    Erythromycin is a naturally-occurringmacrolide derived from Streptomyces

    erythreus

    Structural derivatives include clarithromycinand azithromycin:

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    Macrolide Structure

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    Macrolide binding in thecrytallographic structure ofH.

    marismortui50S ribosomal subunit

    http://www.ncbi.nlm.nih.gov/pubmed/11698379

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    Aminoglycosides

    Initial discovery in the late 1940s, withstreptomycin being the first used; gentamicin,tobramycin and amikacin are most commonly usedaminoglycosides

    All derived from an actinomycete or aresemisynthetic derivatives

    Consist of 2 or more amino sugars linked to anaminocyclitol ring by glycosidic bonds =aminoglycoside

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    Aminoglycoside Structure

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    AminoglycosidesMechanism of Action

    Multifactorial, but ultimately involvesinhibition of protein synthesis

    Irreversibly bind to 30S ribosomesdisrupting the initiation of proteinsynthesis, decreases overall proteinsynthesis, and produces misreading of

    mRNA Most are bacteriocidal

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    Streptogramins

    Example: Synercid synthetic pristinamycin derivatives in a

    30:70 w/w ratio - Quinupristin:Dalfopristin

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    Linezolid

    Mechanism of Action

    Binds to the 50S ribosomal subunit near tosurface interface of 30S subunit causes

    inhibition of 70S initiation complex whichinhibits protein synthesis

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    Clindamycin

    Clindamycin is a semisynthetic derivative of

    lincomycin which was isolated from

    Streptomyces lincolnesis in 1962

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    Clindamycin

    Mechanism of Action

    Inhibits protein synthesis by bindingexclusively to the 50S ribosomal subunit

    Binds in close proximity to macrolides competitive inhibition

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    Tetracycline

    Tetracyclines inhibitbacterial protein

    synthesis by blocking

    the attachment of thetransfer RNA-amino

    acid to the ribosome.

    More precisely they

    are inhibitors of thecodon-anticodon

    interaction.

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    Metronidazole

    Metronidazole is a synthetic nitroimidazoleantibiotic derived from azomycin. First found

    to be active against protozoa, and then

    against anaerobes where it is still extremelyuseful.

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    Metronidazole

    Mechanism of Action

    Ultimately inhibits DNA synthesis Selective toxicity against anaerobic and

    microaerophilic bacteria due to the presence offerredoxins within these bacteria

    Ferredoxins donate electrons to form highlyreactive nitro anion which damage bacterial

    DNA and cause cell death

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    Summary of antibiotic action on the

    genetic mechanism

    http://hstalks.com/main/view_talk.php?t=798&r=285&c=252

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    Mutation and genetic

    disorders

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    Mutation

    Hereditable change in DNA resulting from change innucleotide sequence

    Types of mutation Point mutations

    Swap one base for another; insert a base; delete a base

    MacrolesionsAlteration of the number of copies of a short repeat and

    Large insertions into a gene Multiple causes

    SpontaneousDNA replication/repair errors Insertion of transposons

    Mutagens (physical, chemical, viral)

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    DNA Mutations: An Overview

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    Point Mutations Single or few base pair changes Provide background rate of mutation

    critically important to evolution More likely to lead to loss of function than gain of

    function

    Causes point mutation Induced action of mutagen; environmental agent that alters nucleotide

    sequence

    process of inducing mutations by mutagens called mutagenesis

    Spontaneous arise in absence of known mutagen

    may be due to tautomerism may be caused by errors in DNA replication

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    Types of point mutation

    Base substitution transition

    A G (purine purine) (AT GC) C T (pyrimidine pyrimidine) (CG TA)

    transversion purine pyrimidine (e.g., A C) (AT CG)

    Addition or deletion of nucleotide pairs (base-pairaddition or deletion) also called indelmutations

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    Types of mutation and effects

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    Functional consequences

    Missense mutations differ in severity conservative AA substitution substitutes chemicallysimilar AA; less likely to alter function

    nonconservative AA substitution replaces chemicallydifferent AA; more likely to alter function

    Nonsense mutation results in premature terminationof translation

    truncated polypeptides often are nonfunctional Point mutation in non-coding region may or may not

    have visible effect (e.g., regulatory region)

    S t M t ti

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    Spontaneous Mutation:

    Tautomeric shift

    Natural variation in chemical form of base(isomers); differ in position of atoms & bonds amino (normal) imino (rare) keto (normal) enol (rare)

    Results in mutation during DNA replication base in rare tautomeric form pairs with chemicallysimilar base of its normal complement

    e.g., CG C*G at replication results in C*A whichresolves to CA upon reverse of tautomeric shift

    at next DNA replication event, use of A strand resultsin TA base pair, a transition

    Contributes to spontaneous mutation rate

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    Common Tautomers & Base-

    pairingThe more common keto and amino form of eachbase

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    Rare tautomers

    base-pair differentlythan the morecommon tautomers

    T(enol) pairs with G

    G(enol) pairs with T

    C(imino) pairs with A

    A(imino) pairs with C

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    Tautomeric shifts can be a source of rare spontaneous mutation

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    During the next round of DNA replication, the transitionmutation becomes fixed in the DNA.

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    Creation of indel mutations

    The slippage model for creation of insertions and deletions

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    Induced mutation: Physical Agents

    Ultraviolet radiationThymine is most susceptible; UV radiation

    generates thymine diradicals which can

    dimerizes (pyrimidine-pyrimidine dimers)activates SOS system, resulting in insertion of

    incorrect base

    Ionizing radiationGenerates free radicals from cleavage of

    molecules including H2O, bases, sugar-phosphate backbones

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    Formation of the most toxic and mutagenic DNA lesion, cyclobutane-pyrimidine dimers by UV radiation.

    Dimers can form between two adjacent pyrimidines. (A) thymine-thyminecyclobutane-pyrimidine dimer, and (B) thymine-cytosine dimer and their

    photoreactivation by the enzyme photolyase in the presence of light.

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    UV light inducesthe formation ofcyclobutane

    dimers(pyrimidinedimers) and 6-4photoproducts

    These products

    cause significantkinks in theDNA and stopreplication andtranscription

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    Free-radical effects

    Oxidative damage to basescaused by superoxide and peroxide radicalschemically alter base pairing properties

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    Oxidatively Damaged Bases

    Active oxygen species like superoxide radicals (O2), hydrogenperoxide (H2O2) and hydroxyl radicals (OH

    .) are produced byaerobic respiration and can damage DNA

    Pairs with A

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    Induced Mutation: Chemical agents

    Alkylating agents Intercalating agentsDeaminating agents

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    Alkylating Agents

    Depurination, spontaneous loss of G or A agent modifies base resulting in altered

    pairing e.g., EMS (ethyl methanesulfonate) and NG

    (nitrosoguanidine), formaldehyde, CHCl3, epoxides

    results in replication block and insertion ofnonspecific bases by SOS system

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    Depurination occursmore often than youmight think

    Mammalian cell losesabout 10,000 purinesfrom its DNA in a 20hour cell generationperiod

    Obviously, theselesions must berepaired

    Spontaneous Depurination

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    Alkylating agent: substitutions

    Mutagens have different mutationalspecificity

    Base analogs similar to nitrogenous bases of DNA, but have

    altered pairing properties

    e.g., 5-bromouracil (5-BU) and 2-aminopurine (2-AP) result in transitions

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    Action of 5-BU5-BU can frequentlychange to either the enol or ionized form

    Causes G-C to A-T or A-T to G-C transitions

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    Action of 2-APSimilar to 5-BU but with a different specificity

    A-T to G-C transitions and G-C to A-T transitions

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    Intercalating agents

    flat, planar molecules intercalate betweenbase pairs, disrupt DNA synthesis

    e.g., proflavin, acridine orange, aflatoxin,benzo(a)pyrene

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    Intercalating agentscan cause single baseadditions and deletions

    R ti I t l ti

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    Reactive Intercalation:

    Aflatoxin

    Aflatoxin modifies guanine andresults in lossof base in DNA

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    Deamination

    Deamination, converts cytosine to uracilwhich pairs with adenine at replication

    Indel mutationsresult in translation frameshiftoften occur in regions of repeated bases

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    Deamination of Cyields U whichbase pairs with A

    Results in C-G toT-A transition

    This occurs

    spontaneously

    Deamination

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    Hotspots for mutation often reflect an aspect of the DNAsequence, for example, a sequence repeat

    FS5, FS25, FS45, FS65 result from addition of one set of CTGG which isrepeated three times in the lacIgene

    FS2, FS84 result from loss of one set of CTGG

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    The Ames test was developedby Bruce Ames for evaluatingvarious chemicals for theirpotential as mutagens andcarcinogens

    The Ames Test

    1. Chemical injected into

    mouse where it is metabolized

    2. Liver (site of most chemicalmetabolism) is isolated and anextract is made that containsthe metabolized chemical

    3. Extract is tested for mutagenicityin 3 strains of Salmonellato score forvarious kinds of mutations

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    Defense mechanisms

    Free radical scavengersVitamins A, E, C; only C is water soluble and

    does not accumulate in the body

    GSH (glutathione) converted to GSSGMetallothionein (61 amino acids with 20

    cysteines and 7 zinc atoms

    EnzymesPhotolyase: photoreactivation repair

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    Photoreactivation repair

    Various enzymes with specific properties,e.g. cleavage of pyrimidine dimers (T^T)

    enzyme binds to dimer

    energy of blue light cleaves bond

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    Defense mechanisms: enzymes

    Demethylase (suicide enzyme)Superoxide dismutase (converts superoxides

    to H2O2)

    Catalase (converts H2O2 to H2O + O2Peroxidases

    Repair systems

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    Defense mechanisms: DNA Repair

    DNA damage must be repaired Damaged DNA in any cell will be deleterious to

    cellular function

    in germ line cells mutations heritableBacteria are particularly prone to damage

    haploid all mutations are dominant

    unicellular whole organism affected

    Repair most effective before replication at site Repair can be more or less error-prone

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    Types of Repair: Mismatch

    Mismatch repair genes: mut Mismatched bp detected Nearby ss cut and excision of ssDNA

    past mismatch

    daughter strand preferentially excised DNA polymerase repairs gap

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    Apurinic gap repair

    Apurinic gaps most common spontaneous degradation of

    DNA

    hydrolysis of sugar-purine bond - loss of purine(A, G)

    Repair by AP endonuclease removes base, ss gap filled by polymerase

    If no repair then A inserted at replication G-C T-A transversion mutation

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    Excision repair

    Multi-enzyme system Repairs stretch of damaged nts

    damaged ssDNA excised

    gap repair by polymerase

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    Nucleotide-excision

    repair removes bulky

    DNA lesions that

    distort the doublehelix

    http://www.nature.com/

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    Post-replication repair

    Polymerase cant replicate acrossdamaged nts Leaves a ss gap Gap filled by strand exchange from the

    other dsDNA

    Secondary gap repaired by polymerase